PythonProgrammingonPCRPrimersDesign-CalStateLA.pptVIP

Python Programming on PCR Primers Design Ronny Chan SoCalBSI July 28, 2004 Polymerase Chain Reaction PCR is a technique that is used to amplify a sample of DNA from miniscule amount of DNA (ex., DNA from a crime scene, archaeological samples, organisms that can’t be cultured). Who developed PCR? PCR was developed by Kary Mullis. Kary Mullis is a scientist and surfer from Newport Beach, California. He won a Nobel Prize in Chemistry in 1993 for the development of PCR. He was working for Cetus Corporation in the 70’s and received $10,000 bonus for the idea. How is PCR used? Medical Diagnosis: To detect and identify the causes of infectious diseases from bacteria and viruses. Genetic testing: To determine whether a genetic mutation has been passed on (ex. cystic fibrosis). Evolutionary study: To gather archaeological samples and analyzed for similarities/differences. DNA fingerprinting: To profile DNA from blood, hair, and skin cells for criminal identification and forensics Stages of PCR What is a primer? A primer is a short oligonucleotide which is the reverse complement of a region of a DNA template. It would anneal to a DNA strand to facilitate the amplification of the targeted DNA sequence. Primer Selection variables Primer length Melting Temperature GC content Hair-pin loop Self-dimerization Cross-dimerization Primer Length Should be between 18 – 25 bases. The longer the primer, the more inefficient the annealing. If primers are too short, they will cause non-specific annealing and end up amplifying non-specific sequences. Melting Temperature Formula (18-25 bp range): Tm = 2(A+T) + 4(G+C) The forward and reverse primers should be having similar Tm, or else amplification will be less efficient. Melting Temperature should be between 55oC and 65oC. GC Content GC% = (G + C) / length of seq * 100% The base composition should be in the range of 45% to 55%. Poly G’s or C’s can result in non-specific annealing. Hairpin Loop Self-dimerization Cross Dimerization Forwar